Ion channels in mammalian systems have been, and currently are, the subject of intensive scientific investigation because of the importance and variety of their biochemical functions. Ion channels are now understood to be polypeptide or protein structures with tertiary-quaternary structure forming interior pores embedded in cell membrane walls, that control the flow of ionic currents.
There are many types of ion channels which share both similarity of function and amino acid sequence, thus defining familial relationships between many of these channels. Current work shows there are ion channel families comprised of voltage gated sodium, potassium, and calcium channels, as well as the ligand gated acetylcholine receptors, glycine receptors, and gamma aminobutyric acid receptors.
A great deal is known about voltage gated sodium channels. These are transmembrane proteins responsible for the early sodium permeability increase underlying initial depolarization of the action potential in many excitable cells such as muscle, nerve, and cardiac cells. However knowledge of non-voltage gated sodium channels that are involved in either determining resting membrane potential in the brain or in responding to neurotransmitters is virtually nonexistent.
This is despite the fact that several brain diseases have been associated with channel abnormalities and central nervous system dysfunction. Psychiatric diseases including depression and schizophrenia, and dementias, such as Alzheimer's all have association with dysfunction of the central nervous system whose neurons are controlled and regulated by sodium channels.
Considerably more work has been accomplished with voltage dependent sodium channels. The molecular characteristics of these channels has proven quite complex with multiple isoforms, differential tissue expression and limited sequence conservation between the various families of proteins.
Recent studies have identified a new family of Na+ channels whose characteristic features include Na+ selectivity, inhibition by amiloride, and a conserved primary structure (Chalfie, M., (1990) Nature 345, 410-416; Driscol, M., (1991) Nature 349, 588-593; Huang, M., (1994) Nature 367, 467-470; Canessa, C. M., (1993) Nature 361, 467-470; Canessa, C. M., (1994) Nature 367, 463-467; McDonald, F. J., (1994) Am. J. Physiol. 266, L728-L734; McDonald, F. J., (1995) Am. J. Physiol. 268, C1157-C1163; Voilley, N., (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 247-251; Lingueglia, E. (1993) FEBS Lett. 318, 95-99; Waldmann, R. (1995) J. Biol Chem. 270, 27411-27414; Lingueglia, E. (1995) Nature 378, 730-733). Family members contain 500 to 800 residues. Sequence analysis and studies of topology suggest that the amino and carboxyl termini are intracellular, that there are two hydrophobic regions that traverse the membrane (M1 and M2), and that between M1 and M2 there lies a large cysteine-rich extracellular domain (Snyder, P. M. (1994) J. Biol. Chem. 269, 24379.congruent.24383; Renard, S. (1994) J. Biol. Chem. 269, 12981-12986; Canessa, C. M. (1994) Am. J. Physiol. 267, C1682-C1690).
The best characterized members of this family are the amiloride-sensitive epithelial Na+ channels (ENaC) that control Na+ and fluid absorption in the kidney, colon, and lung. ENaC channels are constructed from at least three homologous subunits (.alpha.-, .beta.-, and .gamma.ENaC) (Canessa, C. M., (1993) Nature 361, 467-470; Canessa, C. M., (1994) Nature 367, 463-467; McDonald, F. J., (1994) Am. J. Physiol. 266, L728-L734; McDonald, F. J., (1995) Am. J. Physiol. 268, C1157-C1163; Voilley, N., (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 247-251; Lingueglia, E. (1993) FEBS Lett. :318, 95-99). Mutations in this channel cause a hereditary form of hypertension called Liddle's syndrome (Shimkets, R. A., (1994) Cell 79, 407-414) and pseudohypoaldosteronism (Chang, S. S., (1996) Nature Genetics 12, 248-253). These channels may also be involved in detection of salty taste (Li, X. J. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 1814-1818). A closely related subunit, .delta.NaCh, is expressed in pancreas, testis, ovary, and brain. .delta. NaCh generates Na+channels when coexpressed with .beta.- and .gamma.ENaC (Waldmann, R. (1995) J. Biol Chem. 270, 27411-27414), suggesting that it may be part of the ENaC subfamily of channels. Several family members have also been discovered in C. elegans, including MEC-4, MEC-10, and DEG-1, which when mutated produce a touch-insensitive phenotype (Chalfie, M., (1990) Nature 345, 410-416; Driscol, M., (1991) Nature 349, 588-593; Huang, M., (1994) Nature 367, 467-470). Specific mutations in the C-elegans group cause neural degeneration (Chalfie, M., (1990) Nature 345, 410-416; Driscol, M., (1991) Nature 349, 588-593). Based on this ability to produce cell degeneration, family members in C. elegans are called "degenerins." The most recent addition to this family is a Phe-Met-Arg-Phe-NH.sub.2 (FMRF-amide)-stimulated Na+ channel (FaNaCh) cloned from Helix(Lingueglia, E. (1995) Nature 378, 730-733).
As can be seen from the foregoing a continuing need exits in the art for further identification and characterization of sodium channel proteins to genetically link diseases to mutations in this gene, to identify disease-causing mutations in the gene, for uses as a diagnostic tool to screen populations for a predisposition to brain diseases, to assay for new ligands and antagonists for the channel, to treat brain disease or the enhance brain function, to use for gene therapy protocols for treatment of brain disease, and to further identify and characterize still other novel and closely related members of this subfamily of sodium channels.